Increased frequency of regulatory T Cells and selection of highly potent CD62L+ cells during...

ORIGINAL ARTICLE

Increased frequency of regulatory T Cells and selectionof highly potent CD62L+ cells during treatment of humanlung transplant recipients with rapamycinChristian M. Lange,1* Thuy Yen Vy Tran,1* Harald Farnik,1 Sven Jungblut,2 Torsten Born,1

Thomas O. Wagner1 and Tim O. Hirche2

1 Department of Internal Medicine I, University Hospital, Frankfurt am Main, Germany

2 Department of Pulmonary Medicine, German Clinic for Diagnostics (DKD), Wiesbaden, Germany

Introduction

In the last decades, highly potent immunosuppressive

agents have been developed, which enabled substantial

progress in human organ transplantation. Cyclosporin A

(CsA) and tacrolimus (FK506) are potent inhibitors of

calcineurin, which under normal circumstances induces

the transcription of interleukin-2 (IL-2). Unlike the calci-

neurin inhibitors, that affect the first phase of the T-lym-

phocyte activation, rapamycin (RPM, syn. sirolimus)

affects the second phase, namely the signal transduction

and clonal proliferation of T lymphocytes. The reduction

of lymphokine production and interleukin release by

above immunosuppressant agents lead to reduced func-

tion of effector T cells (Teff), which in turn prevents

acute allograft rejection and markedly improves rates of

short-term allograft survival [1]. However, long-term

application of the currently recommended immunosup-

pressive regimens is burdened with severe side-effects and

their effect on chronic allograft rejection is poor; both

reasons are responsible for the disappointing long-term

results after allograft transplantation [2]. Human lung

transplant recipients are in particular affected by these

issues, because the high immunogenicity of the lung

Keywords

cyclosporin A, lung transplant, lymphocytes,

rapamune, tacrolimus, Treg.

Correspondence

Dr. med. Christian M. Lange, Department of

Internal Medicine I, University Hospital,

60590 Frankfurt am Main, Germany. Tel.:

+49 (0)69 6301 6336; fax: +49 (0)69 6301

6335; e-mail: [email protected]

*These authors contributed equally to this

work.

Received: 10 May 2009

Revision requested: 9 June 2009

Accepted: 1 September 2009

doi:10.1111/j.1432-2277.2009.00973.x

Summary

The currently available immunosuppressive agents applied in human transplan-

tation medicine are highly potent in the protection from acute allograft rejec-

tion. However, long-term allograft survival is still poor as these drugs fail to

sufficiently prevent chronic allograft rejection. Naturally occurring regulatory

T cells have been postulated as the key players to establish long-lasting trans-

plantation tolerance. Thus, the development of immunosuppressive regimens

which shift the pathological balance of cytopathic versus regulatory T cells of

human allograft recipients towards a protective T-cell composition is a promis-

ing approach to overcome limitations of current transplantation medicine.

Thirty-three patients that received rapamycin (RPM) or calcineurin inhibitor

treatment following lung transplantation were included and their T-cell com-

partments analysed. Twelve healthy volunteers without history of lung disease

served as controls. In this article, we show that treatment of human lung trans-

plant recipients with RPM is associated with an increased frequency of regula-

tory T cells, as compared with treatment with calcineurin inhibitors or to

healthy controls. Moreover, regulatory T cells during treatment with RPM were

CD62Lhigh, a phenotype that displayed an enhanced immunosuppressive

capacity ex vivo. Our data support the use of RPM in human lung transplant

recipients and undertaking of further prospective studies evaluating its impact

on allograft and patient survival.

Transplant International ISSN 0934-0874

ª 2009 The Authors

266 Journal compilation ª 2009 European Society for Organ Transplantation 23 (2010) 266–276

requires a comparably aggressive immunosuppression [2].

In view of these issues, there is strong need for improved

immunosuppressive strategies that can establish specific

transplantation tolerance, without continual general

immunosuppression.

The development of immunosuppressive regimens

which shift the pathological balance of cytopathic versus

regulatory T cells (Tregs) of human allograft recipients

towards a protective T-cell composition is a promising

approach to overcome limitations of current transplanta-

tion medicine. Recent studies in animal models have

confirmed that CD4+CD25+Foxp3+ Tregs are crucial to

sustain ongoing transplant tolerance [3,4]. For example, it

was shown that the transfer of Tregs alone without any

additional immunosuppressive agents is sufficient to

avoid solid allograft rejection in animal models [3].

Initially, Tregs were thought to exclusively develop in the

thymus through positive selection. However, more recent

studies suggest that Tregs can also develop in peripheral

compartments from naı̈ve CD4+ T cells in an antigen-

specific manner, mediated by peptide-presenting dendritic

cells [4–7]. This is an important prerequisite for the

development of allograft-specific Tregs, as the thymus

does not contain any allo-antigen. Studies in animal

models and in vitro revealed that different immunosup-

pressive drugs can promote or abrogate de novo genera-

tion of Tregs. For example, RPM was shown to support

the de novo generation of Tregs in the periphery of mice

after skin transplantation [8]. In contrast, application of

CsA resulted in a strong reduction of the peripheral Treg

compartment in mice.

As up to this point, the role of Tregs in human allograft

recipients still needs to be better characterized. The fre-

quency of Tregs in the circulation and in biopsy specimens

of renal and lung allograft recipients has been correlated

positively with allograft survival [9–13]. Accordingly, acute

liver rejection was shown to be associated with decreased

numbers of Tregs [14]. However, as studies evaluating

Tregs and Teffs in human allograft recipients under differ-

ent immunosuppressive regimens are rare, more studies

are necessary to translate these convincing experimental

data into clinical practice.

In this study, we aimed to characterize the effect of

currently recommended immunosuppressive regimes on

the frequency and function of Tregs in human lung trans-

plant recipients. In this study, we show for the first time

that treatment of human lung transplant recipients with

RPM in combination with mycophenolate mofetil (MMF)

is associated with an increased frequency of Tregs, as

compared with treatment with calcineurin inhibitors or

to healthy control individuals. Moreover, during treat-

ment with RPM/MMF, Tregs were CD62Lhigh, a pheno-

type that displayed an enhanced immunosuppressive

capacity ex vivo. In contrast, alterations in the phenotype

of Teff during immunosuppression, namely a shift

towards Th2 cell predominance (Pred), were independent

of the applied compounds.

Materials and methods

Patients and clinical samples

Thirty-three lung transplant recipients (mean age:

50.6 years, range 21–74 years) were selected from lung

transplants performed at the University Hospital Frank-

furt, Germany, between 1986 and 2006 according to the

following inclusion criteria: post-transplant follow up for

at least 12 months, stable clinical condition without any

signs of infection or acute allograft rejection at time of

enrolment, treatment with the same immunosuppressive

regimen for at least 4 months prior to enrolment. Patients

were divided into three groups according to their immu-

nosuppressive regimen (CsA, n = 14; FK506, n = 12;

RPM, n = 7). All patients additionally received MMF and

low-dose Pred (5–10 mg/day p.o.). Plasma concentrations

of immunosuppressive drugs were: cyclosporine 120–

180 ng/ml, tacrolimus 6–8 ng/ml, rapamycin 6–8 ng/ml

and MMF 1.5–3 ng/ml. Treatment indication for rapamy-

cin was a reduction of nephrotoxicity during immunosup-

pressive therapy in all seven patients. Twelve healthy

volunteers without a previous history of lung disease

served as controls. Demographic and clinical data are

summarized in Table 1. Peripheral ethylene diamine tetra-

acetic acid (EDTA)-blood was taken from all patients and

healthy controls by peripheral vein puncture. Institutional

board approval and informed consent from all patients

was obtained in accordance with the Helsinki declaration.

Reagents and antibodies

For flow cytometry, the following antihuman monoclonal

antibodies were used (all fluorochrome-conjugated):

anti-CD4-fluorescein isothiocyanate (FITC), anti-CD4-

PercP, anti-CD4-APC, anti-CD25-phycoerythrin (PE),

anti-CD25-FITC, anti-CD62L-FITC, anti-CD127-PE, anti-

ICOS-PE, anti-BTLA-PE (all from Pharmingen, Heidelberg,

Germany), anti-Foxp3-PE, anti-Foxp3-APC, anti-IFN-

gamma-PE, anti-IL-4-PE (all from Miltenyi Biotec, Bergisch

Gladbach, Germany) and anti-Tim3-PE (RD Systems,

Wiesbaden, Germany). Appropriate immunoglobulin

isotypes were used as a control. Cytofix/Cytoperm and

Wash/Perm for intracellular cytokine staining were from BD

Biosciences (Heidelberg, Germany). CFSE was obtained

from Invitrogen (Karlsruhe, Germany). Magnetic cell

separation was performed with commercially available kits

for isolation of CD4+CD25+ Tregs, CD4+ T cells, naı̈ve

CD4+ T cells, and CD62L T cells (all from Miltenyi Biotec).

Lange et al. Rapamune increases Treg lymphocyte compartment in human lung transplant recipients

ª 2009 The Authors

Journal compilation ª 2009 European Society for Organ Transplantation 23 (2010) 266–276 267

For in vitro T-cell stimulation, a T-cell activation/expansion

kit was used, which is based on beads coated with anti-CD3

and anti-CD28 from Miltenyi Biotec. For in vitro experi-

ments the following immunosuppressive agents were used:

Cyclosporine A (CsA; Novartis Pharma, Basel, Switzerland),

tacrolimus (FK506; Astellas, Munchen, Germany) and

rapamycin (RPM; Wyeth Laboratories, Philadelphia, PA,

USA).Except where indicated, all other chemicals were

reagent-grade and purchased from Sigma Aldrich (St. Louis,

MO, USA).

Fluorescence staining and flow cytometry

Lymphocytes from freshly drawn peripheral EDTA-blood

were isolated by Histopaque gradient and used immedi-

ately. To determine the absolute cell numbers of the

indicated lymphocyte populations the totality of all live

lymphocytes was counted first by using the trypan blue

method. By using these total cell numbers of all lympho-

cytes the absolute cell numbers of the indicated subpopula-

tions were calculated based on their percentages on

all events during flow cytometric analysis. Fc-receptor

blocking was performed with human IgG (Sigma Aldrich).

For staining of extracellular antigens, approx. 105 cells were

incubated for 20 min with the indicated antibody at room

temperature in the dark, washed twice with PBS containing

1% bovine serum albumin and re-suspended in 20 ll

buffer. For staining of intracellular antigens, approx. 105

cells were first stained for extracellular antigens, followed

by fixation and permeabilization with Cytofix/Cytoperm

for 20 min at room temperature. Then, cells were washed

twice with Wash/Perm and incubated with anti-cytokine

antibodies or anti-FoxP3 antibodies in 20 ll Wash/Perm

for 30 min at room temperature. All antibodies were

titrated for optimal concentrations. Then cells were washed

twice, resolved in 50 ll PBS and analysed by flow cytome-

try. Fluorescence-activated cell sorting was performed on

live cells with a FacsCalibur System (Becton Dickinson,

Heidelberg, Germany) and analysed with Summit software

(v4.1; Dakocytomation, Freiburg, Germany).

T-cell stimulation and ex vivo exposure

to immunosuppressive agents

For ex vivo experiments, naı̈ve CD4+ T cells were isolated

from freshly drawn peripheral EDTA-blood of healthy

volunteers (n = 3 each) by magnetic cell separation

(MACS)-technology, according to the manufacturer’s

Table 1. Demographic and baseline characteristics of study population.

Cyclosporine Tacrolimus Rapamycin Control P-value

Gender, n (%)

Female 8 7 5 6 NS

Male 6 5 2 6

Age, years

Median 51 64 55 46 NS

Range 21–72 23–74 29–67 22–65

BMI, kg/m2

Median 20.9 22.9 20.7 20.7 NS

Range 15.3–28.6 15.2–32.8 15.0–34.0 16.3–27.8

CRP, mg/dl

Median 0.1 0.1 0.2 ND NS

Range 0.02–1.08 0.07–1.82 0.09–1.2

Blood leukocytes, per nl

Median 7.14 7.72 10.0 ND NS

Range 3.0–11.3 4.5–13.0 5–13.7

Underlying disease

CF 7 4 3 NS

COPD 5 7 4

UIP 2 1 0

Years after LTX

Median 4 6 7 NS

Range 1.0–20 2.0–12 2.0–8

BMI, body mass index; CRP, C-reactive protein; CF, cystic fibrosis; COPD, chronic obstructive pulmonary disease; UIP, ususal interstitial pneumoni-

tis; LTX, lung transplantation; NS, not significant; ND, not determined.

Plasma concentrations of immunosuppressive drugs were: cyclosporine 120–180 ng/ml, tacrolimus 6–8 ng/ml, rapamycin 6–8 ng/ml and MMF

1.5–3 ng/ml.

P-value indicates statistical significance between immunosuppressive treatment arms.

Rapamune increases Treg lymphocyte compartment in human lung transplant recipients Lange et al.

ª 2009 The Authors


instructions (Miltenyi Biotec). Then 104 CD4+ T cells

were stimulated by using the above described MACS

T-cell activation/expansion kit following the manufac-

turer’s recommendations. Briefly, cells were cultured in

96-well plates in 200 ll Iscove’s modified Dulbecco’s

medium (IMDM) supplemented with 10% fetal calf

serum in an atmosphere containing 5% CO2. Concentra-

tions of anti-CD3/CD28-beads were chosen and titrated

according to the manufacturer’s instructions. Immuno-

suppressive agents were added at day 0 at the following

concentrations (final) [15]: CsA, 100 ng/ml; FK506,

10 ng/ml; RPM, 10 and 100 ng/ml (as indicated). Follow-

ing incubation cells were washed twice and processed for

flow cytometric analysis of Foxp3, CD25 and CD62L as

described above. Other aliquots were analysed for cell

proliferation and apoptosis as described below.

Treg suppression assay

Suppressive function of Tregs was assessed by using an in

vitro suppression assay. Direct ex vivo isolation of the

indicated T-cell populations was performed by using

MACS-isolation kits from Miltenyi Biotec according to

the manufacturer’s instructions. Freshly isolated

CD4+CD25) T cells (responders) from the blood of lung

transplant recipients and healthy donors (n = 3 each)

were stained with CFSE for 20 min and then washed four

times. Anti-CD3/anti-CD28 coated beads were used as a

stimulus (Miltenyi Biotec). Cell culture was performed in

96-well plates in 200 ll IMDM supplemented with 10%

fetal calf serum in an atmosphere containing 5% CO2.

CD4+CD25) T cells (104) were stimulated for 2 h. Next,

the indicated numbers of CD4+CD25+CD62L) or

CD4+CD25+CD62L+ T cells were added. To separate

CD4+CD25+CD62L+ cells from CD4+CD25+CD62L)cells, washed lymphocytes were selected or depleted with

regard to their expression of CD62L by using CD62L

MACS-isolation kit and subsequently purified by using

the CD4+CD25+ Treg MACS-isolation kit. After 72 h,

the percentage of proliferating responder cells as reflected

by CFSE dilution was assessed by flow cytometry.

Apoptosis assay

Lymphocytes were harvested as described above and

apoptosis was quantified by Annexin V staining using a

kit (Miltenyi Biotec). Briefly, lymphocytes (106) were

washed twice with Annexin V binding buffer, resuspended

in 100 ll binding buffer and stained with 10 ll Annexin

V according to the manufacturer’s instructions. Then,

cells were incubated for 15 min at room temperature,

washed again twice and analysed immediately by flow

cytometry.

Statistics

Data are presented in text and tables in terms of frequen-

cies and means with standard deviation and range, as

appropriate. Differences between subgroups were statisti-

cally compared by Student’s t-test. The general level for

assessment of statistical significance was predefined as

2a = 0.05 (two-sided). Resulting P-values were presented

descriptively without further adjustment for multiple test-

ing. All statistical calculations were performed using sta-

tistical Analysis (sas) software, version 9.1 (SAS Institute

Inc., Cary, NC, USA).

Results

Number, phenotype and cytokine production of

effector CD4+ T cells do not differ among different

immunosuppressive regimes

The influence of the different immunosuppressive regi-

mens on the size of potentially allo-protective and allo-

destructive CD4+ T cell compartments in the peripheral

blood of human lung transplant recipients was quantified

by flow cytometry. Absolute numbers of lymphocytes

were substantially decreased in human lung transplant

recipients (mean 1.5–1.9/nl, P < 0.05), compared to

healthy control individuals (4.3/nl) (Fig. 1a). Similar find-

ings were made when the subgroup of CD4+ T cells was

analysed (0.39–0.57/nl vs. controls 1.7/nl, respectively,

P < 0.05) (Fig. 1b). However, there was no difference for

both lymphocyte and CD4+ T-cell counts among the

immunosuppressive treatment arms (P > 0.05).

To assess whether different immunosuppressive treat-

ment regimes are associated with phenotypic alterations

of circulating Teff, expression of the signature cytokines

IFN-c and IL-4, as well as of negative and positive

co-stimulatory molecules was determined in CD4+ T cells

by flow cytometry. The expression of IFN-c was reduced

in human lung transplant recipients in all treatment arms

when compared to the control group (10.4–12.7% vs.

18.0% respectively) (Fig. 1c). In contrast, the expression

of IL-4 was markedly increased in all patient groups

when compared to the control group (16.5–22.1% vs.

9.2%, respectively, P < 0.05) (Fig. 1d). Thus, in human

lung transplant recipients the proportions of Th1 and

Th2 cells are changed towards a Th2-phenotype during

immunosuppressive therapy, independently of the applied

agents.

The expression of the positive co-stimulatory molecule

Tim3 was significantly reduced in allograft recipients

compared to the control group (9.7–15.1% vs. 23.8%

respectively) (Fig. 2a). Comparable results were obtained

for the expression of the inducible T-cell co-stimulator

ICOS (allograft recipients 4.92–6.25% vs. controls 13.5%,


ª 2009 The Authors


respectively, P < 0.05) (Fig. 2b). In contrast, the inhibi-

tory co-stimulator BTLA was strongly expressed on

peripheral CD4+ T cells during immunosuppressive ther-

apy compared to the control group (76.8–81.1% vs.

50.9%, respectively, P < 0.05) (Fig. 2c). Of note, the

expression of the positive (Tim3, ICOS) and the inhibi-

tory (BTLA) co-stimulants between immunosuppressive

regimens was comparable (P > 0.05). Thus, Teff predomi-

nantly express negative co-stimulatory molecules during

immunosuppression with both calcineurin inhibitor- and

RPM-based combination therapies.

Increased numbers of regulatory T cells following

RPM therapy

To investigate the impact of the different immunosup-

pressant regimes on specific immunomodulatory function

of lymphocytes, we analysed the frequency of Tregs. The

absolute number of CD4+CD25+Foxp3+-Tregs in lung

transplant recipients was significantly higher following

treatment with RPM/MMF/Pred (0.10/nl), when com-

pared to calcineurin inhibitor-based therapy with CsA/

MMF/Pred or FK506/MMF/Pred (0.05/nl, P < 0.05, each)

(Fig. 3a). Of note, the absolute numbers of Tregs under

RPM-based therapy was comparable to healthy controls

individuals (0.10/nl). The mean relative number of Tregs

(b)

1.5

2 * (a)

3

4

5 * Cyclosporine

Tacrolimus

(c)

0

0.5

1

CD

4+ T

cel

ls /

nl

0

1

2

Lym

phoc

ytes

/ nl

Rapamycin

Control

19%

Cyclospo rine Cyclospo rine

8%

(d)

15

20

25

20

30

Rapamycin Rapamycin

4% 21%

IL

7%

Contro l

IFN

Contro l

14%

0

5

10

% IF

N–γ

+ c

ells

0

10

% IL

-4+

cel

ls

* 4% 21%

IL -4 IFN -

Figure 1 Immunosuppressive therapy induces a Th2-dominance in

human lung transplant recipients. (a) Indicated immunosuppressive

therapies (+MMF/Pred) substantially decrease total numbers of lym-

phocytes in peripheral blood of lung transplant recipients. (b) Similar

findings were made when the subgroup of CD4+ T cells was analy-

sed. Differences among immunosuppressive agents were not statisti-

cally significant. Decreased expression of IFN-c (c) and increased

expression of IL-4 (d) in peripheral blood CD4+ T cells under immuno-

suppressive therapy. The data demonstrate that all indicated immuno-

suppressive agents induce a shift towards a Th2-dominance.

Exemplary histograms of the indicated cytokines are shown for cyclo-

sporine- and rapamycin-treated patients and healthy individuals (con-

trol). Bars indicate mean ± SEM (*P < 0.05 for differences between

controls versus patients).

Cyclosporine (a)

9%

R5

Control

Rapamycin

7%

40

10

20

30

% T

im3+

cel

ls

0 17%

Tim3

3%

(b) Cyclosporine

15

20

*

0

5

10

% IC

OS

+ c

ells

13%

Control

Rapamycin

2%

ICOS

87%

(c) Cyclosporine

48%

Control

Rapamycin

79%

25

50

75

100

*

0

% B

TLA

+ c

ells

BTLA

R5

R7

R5

R5

R5

R5

R5

R5

Figure 2 Immunosuppressive therapy alters the phenotype of effec-

tor T cells in human lung transplant recipients. (a,b) Indicated immu-

nosuppressive therapies (+MMF/Pred) down-regulate the expression of

positive co-stimulatory molecules Tim3 and ICOS on effector CD4+ T

cells of lung transplant recipients. (c) In contrast, the inhibitory

co-stimulator BTLA is up-regulated. No significant differences in phe-

notype profile were detected among patients treated with different

immunosuppressive regimens. Exemplary histograms of the indicated

effector molecules are shown for cyclosporine- and rapamycin-treated

patients and healthy individuals (control). Bars indicate mean ± SEM

(*P < 0.05 for differences between controls versus patients).


ª 2009 The Authors


on all CD4+ T cells for the RPM regime (17.2%) was sig-

nificantly higher when compared to both calcineurin-

inhibitors (CsA, 10.8; FK506, 11.2%, P < 0.05), as well as

to the control group (6.0%, P < 0.05) (Fig. 3b). Thus,

absolute, but not relative numbers of Tregs are decreased

during combination therapy with calcineurin inhibitors.

In contrast, both absolute and relative numbers of Tregs

are markedly enhanced in human lung transplant recipi-

ents during treatment with combination therapy with

RPM, as compared with treatment with calcineurin inhib-

itor-based therapies. Interestingly, RPM-based therapy

results in increased relative numbers of Tregs as com-

pared with the healthy control individuals. Of note, intra-

individual Treg counts remained stable over time, as

determined by sequential FACS analysis over 5 months in

a subset of patients (n = 6, data not shown).

Induction of Foxp3-expression by RPM ex vivo

To examine the impact of immunosuppressive agents on

differentiation of Tregs, we analysed Foxp3 expression in

naı̈ve polyclonal CD4+ T cells following ex vivo activation

with anti-CD3 and anti-CD28. Activated CD4+ T cells

(a)

Cyclosporine

Tacrolimus

Rapamycin 0.05

0.1

0.15 *

(b)

Control

0

20 * 25

Cyclosporine

10% 9%

Tacrolimus

0

5

10

15

% C

D4+

CD

25+

Fox

p3+

cel

ls

CD

4+ C

D25

+

Fox

p3+

Tre

gs /

nl

8%

Control Rapamycin

18% R5 R5

R5 R5

Cou

nts

Cou

nts

Foxp3

Figure 3 Increased numbers of regulatory T cells following RPM ther-

apy of human lung transplant recipients. Mean absolute numbers of

CD4+CD25+Foxp3+ regulatory T cells (a) and relative numbers of

CD4+CD25+Foxp3+ regulatory T cells of all CD4+ T cells (b) in the

peripheral blood of human lung transplant recipients under the indi-

cated immunosuppressive therapies (+MMF/Pred). During treatment

with RPM, absolute and relative numbers of CD4+CD25+Foxp3+ reg-

ulatory T cells are higher as compared with calcineurin inhibitor-based

therapies and healthy individuals (control). Exemplary histograms of

the CD4+CD25+Foxp3+ cells are shown on the left. Bars indicate

mean ± SEM (*P < 0.05 for differences between RPM versus CsA/

FK506 and controls versus CsA/FK506).

(a) Tacrolimus

17%

11% R7

10%

33%

Control Rapamycin

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

(b)

Foxp3

Tacrolimus Cyclosporine

Cyclosporine

Control Rapamycin

CFSE

(c)

64% 87%

Tacrolimus Cyclosporine

39% 43%

Control Rapamycin

AnnexinV

R7

R7 R7

R4 R4

R4 R4

Figure 4 Foxp3-expression, cell proliferation and apoptosis of CD4+

T cells in the presence of different immunosuppressive agents ex vivo.

Naı̈ve CD4+ T cells were isolated from peripheral blood of healthy vol-

unteers by MACS-sorting and then stimulated with anti-CD3 and anti-

CD28 in the presence of the indicated immunosuppressive agents or

PBS (control). After 3 days of culture, Foxp3-expression (a), cell prolif-

eration (CFSE intensity) (b), and apoptosis (percentage of AnnexinV

positive cells) (c) were determined by flow cytometry. Representative

histograms show that the proportion of Foxp3-expression and rates of

cell proliferation are significantly higher, but the percentage of apop-

totic cells was lower in the presence of RPM as compared with calci-

neurin inhibitors.


ª 2009 The Authors


expressed markedly higher levels of Foxp3 after 3 days of

culture in the presence of RPM (28.1%) compared to cal-

cineurin inhibitors (CsA, 10.3; FK506, 13.4%) or PBS

(10–20%) (Fig. 4a). Equivalent to Foxp3 the majority of

these CD4+Foxp3+ T cells up-regulated CD25 (data not

shown). In parallel, cell proliferation was higher in the

presence of PRM than in the presence of calcineurin

inhibitors, as assessed by carboxyfluorescein diacetate

succinimidyl ester (CFSE)-staining (Fig. 4b). To assess

whether rates of apoptotic cells differ in stimulated CD4+

T cells as a function of the different immunosuppressive

agents used, CD4+ T cells were stained with AnnexinV

after 3 days of cell culture. The percentage of AnnexinV

positive CD4+ T cells was significantly lower when cul-

tured in the presence of RPM (30–45%) compared to cal-

cineurin inhibitors (CsA, 70–90%; FK506, 60–80%)

(Fig. 4c). Of note, the percentage of apoptotic cells fol-

lowing culture with RPM or placebo (30–50%) was com-

parable (P > 0.05).

Regulatory T cells during RPM-based

immunosuppression are CD62Lhigh

Based on the expression of surface receptors, CD4+ Teff

can be further divided into functionally distinct popula-

tions. In some animal models, CD4+CD25+CD62L+

Tregs have been shown to serve as essential suppressors

of autoimmunity [5]. To assess the impact of differential

immunosuppressive therapy on CD62L+ receptor expres-

sion, we quantified CD62L+ concentration on

CD4+CD25+Foxp3+-Tregs by flow cytometry. Following

treatment of patients with RPM/MMF/Pred, large frac-

tions of Tregs were found positive for CD62+ (37.7%). In

contrast, the fractions of Tregs that were positive for

CD62+ were significantly lower following treatment of

patients with either CsA/MMF/Pred (23.2%), FK506/

MMF/Pred (20.4%) or in healthy control individuals

(17.6%), P < 0.05 each (Fig. 5). Thus, RPM-based immu-

nosuppressive therapy is associated with an accumulation

of CD62L+ Tregs in peripheral blood of human lung

transplant recipients.

CD62Lhigh regulatory T cells have increased

immunosuppressive capacity ex vivo

Earlier studies in some animal models have shown

enhanced immunosuppressive capacity of Tregs following

co-incubation with RPM ex vivo [16]. In the light of our

above findings, we postulate that enhanced immunosup-

pressive capacity following RPM treatment is mediated by

an extended compartment of CD62L+ cells. To investigate

the impact of CD62L+ Tregs on immunosuppression, we

isolated CD4+CD25+CD62L+ and CD4+CD25+CD62L)

T cells by MACS from peripheral blood of healthy

individuals and human lung transplant recipients treated

with RPM-based regimen. The obtained cell populations

were at least 80% Foxp3-positive, as quantified by

flow cytometry. Next, the immunosuppressive capacity of

CD4+CD25+CD62L+ and CD4+CD25+CD62L) T cells

was examined by incubation with polyclonal, stimulated,

CFSE-labelled, naı̈ve CD4+CD25) T cells for 72 h. Under

our experimental conditions, the capacity for suppression

of activated Teff was found significantly higher following

incubation with CD62L+ Tregs as compared with

CD62L) Tregs (P < 0.05) (Fig. 6a and b). Of note, the

immunosuppressive capacity of CD62L+ Tregs from

RPM-treated lung transplant recipients and healthy indi-

viduals was equivalent (Fig. 6b and c) (P > 0.05). Thus,

RPM-based immunosuppressive therapy is associated

with the presence of a high proportion of CD62Lhigh

Tregs, which display enhanced immunosuppressive

qualities.

Discussion

In this study, we characterized for the first time the Teff

and Treg compartment in the peripheral blood of human

lung transplant recipients receiving combination therapies

of CsA/MMF/Pred, FK506/MMF/Pred and RPM/MMF/

Pred. In this study, we show that the frequency of Teff is

substantially reduced in all groups as compared with

25

17%

Cyclosporine Tacrollimus

15%

5

10

15

20 * (a)

CD

62L

31%

Rapamycin Control

14%

0

% C

D25

/ C

D62

L+

CD25 CD127

(b) 5%

R5

Figure 5 Regulatory T cells are CD62Lhigh in human lung transplant

recipients treated with RPM. (a) Representative scatter plots (left)

show co-expression of CD25 and CD62L in CD4+Foxp3+ Tregs that

were isolated from the peripheral blood of human lung transplant

recipients under the indicated immunosuppressive therapies (+MMF/

Pred). As compared with calcineurin inhibitor-treated patients and to

healthy individuals (controls), RPM-based immunosuppressive therapy

is associated with increased numbers of CD62Lhigh Tregs. Bars

indicate mean ± SEM (*P < 0.05 for differences between RPM versus

CsA, FK506 and controls). (b) A representative histogram of CD127-

expression on CD4+CD25+CD62L+ T cells is shown. CD4+CD25+

CD62L+ T cells express low levels of CD127.


ª 2009 The Authors


healthy individuals. In contrast, the Treg compartment is

exclusively enlarged in individuals exposed to RPM-based

treatment. Absolute numbers of Tregs under RPM treat-

ment were even slightly higher than in healthy individu-

als. In contrast, CsA- or FK506-based treatment resulted

in marked reduction of the Tregs compartment.

Our data confirm and extend the findings of previous

studies that postulated a differential effect between the

above-mentioned immunosuppressive agents on graft-

destructive Teff and graft-protective Tregs. It was shown

that CsA inhibits, but RPM promotes activation-induced

cell death of Teff [17]. Several studies revealed detrimen-

tal effects of calcineurin inhibitors such as CsA or FK506

on the survival and function of Tregs [18,19], which can

be explained by the calcineurin inhibitor-induced inter-

ruption of IL-2-signalling, a key cytokine in Treg develop-

ment and homeostasis [19]. In contrast, RPM-exposure

can promote de novo generation and gain of Treg func-

tion in vitro [8,18,20,21]. However, as studies evaluating

Tregs and Teff in human allograft recipients under differ-

ent immunosuppressive regimens are rare, these convinc-

ing experimental data have not been translated into

clinical practice so far.

Because all patients in this study received combination

therapies comprising MMF and low-dose Pred, our setting

does not unarguably allow attributing the observed effects

to RPM. However, previous in vitro studies did not find a

significant effect of MMF alone on the Treg compartment

[19]. Pred was shown to promote Treg survival in vitro

and in vivo [22]. In this study, co-administration of MMF

and Pred was similar in all groups and the dosage of Pred

was low in all patients. Thus, RPM seems to play the

pivotal role in the enlargement of the Treg compartment.

As combination regimens are still mandatory in human

lung transplant recipients, characterization of Tregs during

such therapies is of considerable interest and thus reflects

clinical scenarios.

Previous studies in mice have postulated a particular

role of CD62Lhigh Tregs. For example, CD62Lhigh, but

not CD62Llow Tregs were able to inhibit diabetes devel-

opment in nonobese diabetic mice [5]. Importantly, these

CD62Lhigh Tregs could be selectively expanded in vitro

Teg:Teffr

CD25+ CD62L–

CD25+ CD62L+

0.25:1

(a)

0.5:1

1:1

1.5:1

CFSE intensity

2:1

(b)

CD62L+ Treg

CD62L– Treg

*

*

0

20 % c

ycle

d ce

lls

40

60

80

100

0.25 /1

1/ 1

2/ 1

Treg/ Teff

% c

ycle

d ce

lls0

20

40

60

80

100 (c)

*

0.25 /1

1/ 1

2/ 1

Treg/ Teff

CD62L+ Treg

CD62L– Treg

Figure 6 CD62L high regulatory T cells show enhanced immunosuppressive capacity ex vivo. (a) Freshly isolated CD4+CD25) T cells from the

blood of healthy donors were stained with CFSE and stimulated with anti-CD3 and anti-CD28. The indicated ratios of CD62L):CD62L+ Tregs

were added and CFSE-intensity was determined by flow cytometry after 72 h. The capacity for suppression of activated effector T cells was signifi-

cantly higher following incubation with CD62L+ as compared with CD62L) Tregs. Exemplary histograms of the CFSE intensity are shown. (b)

Results of three separate experiments as described in (a) are shown. Results are expressed as the percentage of cycled CFSE+ cells as: (number of

cycled CFSE+ cells/total number of cycled and noncycled cells). (c) Results of three experiments as described in (a) and (b) are shown with the dif-

ference that regulatory and effector T cells were obtained from human lung transplant recipients who were treated with rapamycin. There are no

significant differences in immunosuppressive capacity of Tregs from healthy control individuals or human lung transplant recipients (P > 0.05). Bars

indicate mean ± SEM (*P < 0.05 for differences between suppressive capacity of CD62L) and CD62L+ Tregs).


ª 2009 The Authors


by dendritic cells without losing their phenotype [5]. In

addition, it was shown that CD62Lhigh but not

CD62Llow Tregs are highly potent in suppressing acute

graft-versus-host disease in mice [23].In this study, we

found that the subset of CD62Lhigh Tregs is increased in

human lung transplant recipients during RPM-based

treatment as compared with individuals treated with cal-

cineurin inhibitors and also as compared with healthy

control individuals. Moreover, we observed a superior

immunosuppressive capacity of CD62Lhigh Tregs ex vivo,

as compared with CD62Llow Tregs. These data are the

first, which characterize CD62Lhigh Tregs in human allo-

graft recipients and indicate a favourable immune-modu-

lating effect of RPM. Together with the quantitatively

enlarged Treg compartment, the selection of CD62Lhigh

Tregs during RPM-based immunosuppression is support-

ive of a significantly enhanced immunosuppressive capac-

ity of the Treg pool. As CD62L is an integrin enabling

recirculation through lymphoid tissues, the selection of

CD62Lhigh Tregs might be an essential feature of host

immunity. CD62L expression characterizes memory Teff

and Treg populations, which are determined to home to

the lymph nodes in contrast to inflammation-seeking

CD62Llow T cells [24–26]. Induction of CD62Lhigh

Tregs under RPM-based treatment might inhibit the

priming of naı̈ve allograft-specific T cells in the draining

lymph nodes and thus help to prevent chronic allograft

rejection.

In theory, there are at least two ways for expansion of

the Treg compartment in human allograft recipients:

First, their generation or gain of function could be

induced in vivo by specific compounds. Second, Tregs

could be de novo generated in vitro and transferred to the

recipient thereafter. In vivo, the extrathymic de novo gen-

eration of Tregs is pivotal, because mainly those Tregs are

allo-antigen specific [27]. Whether a naı̈ve T cell differen-

tiates into a Treg or a Teff seems to crucially depend on

the local cytokine milieu. In vitro experiments have

shown that RPM inhibits the production of pro-inflam-

matory cytokines and promotes the expression of TGF-

beta [15,28]. Together with its inhibitory impact on the

cell cycle, RPM might use this pathway to induce the

de novo generation of Treg. Thus, it can be speculated

that the high number of Tregs during RPM-based therapy

is at least partially attributable to peripheral de novo

generation from naı̈ve T cells. In this study, we show that

activation of naive CD4+ T cells in the presence of rapa-

mycin but not of calcineurin inhibitors results in the

induction of Foxp3. This finding might reflect a differen-

tiation of naive T cells to Tregs in the presence of

rapamycin in vitro. However, this cannot be established

definitely with our experiments as a recent study has

shown that Foxp3 can be expressed transiently in Teff as

well [29].

In our study, we did not observe differences in the

Teff compartment during treatment with rapamycin as

compared with calcineurin inhibitors. In all treatment

arms, we report a polarization towards a Th2 cell

phenotype, which is indicated by a reduced expression of

IFN-c and Tim3 on the one hand and an enhanced

expression of IL-4 and BTLA on the other hand. Tim3,

which is predominantly expressed on Th1 cells, seems to

play an important role in the maintenance of immune

tolerance as engagement of Tim3 by its receptor galectin

9 results in apoptosis of Tim3-expressing T cells [30].

The functional consequences of low ICOS expression,

which we have observed in all human lung transplant

recipients, are less clear. ICOS plays a complex role in

adaptive immunity, which is indicated by the observation

that loss of ICOS can result in common variable immu-

nodeficiency [31]. In addition, it was shown that Tregs

within pancreatic islets of diabetic mice critically depend

on ICOS-signalling [32]. Low ICOS-expression during

immunosuppressive therapy might be explained by the

fact that ICOS-expression depends on T-cell activation,

which is abrogated in the presence of immunosuppressive

agents. Because recent data indicate a role of Th17 cells

in allograft rejection, investigating these cells in patients

during treatment with rapamycin is of interest for further

studies [33].

In conclusion, we show that the frequency of highly

immunosuppressive CD62Lhigh Tregs in the peripheral

blood of human lung transplant recipients is significantly

increased during RPM-based combination therapy, as com-

pared with calcineurin inhibitor-based treatment and to

healthy control individuals. Thus, our data suggest that

RPM can be used to preferentially promote Treg-mediated

transplantation tolerance. Prospective studies in human

allograft recipients evaluating potential benefits of RPM-

based therapy on patient and allograft survival are required

to determine the clinical significance of these findings.

Authorship

CML, SJ, TOW and TOH: planning the study. CML, TYT

and HF: collecting the data. CML, TYT and TOH: analy-

sis of data. CML, TYT, HF, SJ, TB, TOW and TOH:

preparation and revision of the manuscript.

Acknowledgements

TOH has received research grants from the Frankfurt

University School of Medicine and the Christiane Herzog

Foundation.


ª 2009 The Authors


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